The following is a discussion of the relevant art, none of which is admitted to be prior art to the appended claims.
Winkler et al. (J. Org. Chem 1983, 48: 3119-3122) disclose the compound 7-deaza-2′-deoxyguanosine.
U.S. Pat. Nos. 4,804,748 and 5,480,980 disclose that 7-deaza-dG derivatives of the family (I): wherein R is PO3H2, P2O6H3, P3O9H4 or an alkali metal, alkaline earth metal, or ammonium salt of the phosphate groups; are useful in sequencing in that they provide good gel electrophoretic separation of guanosine, cytosine-rich sequence fragments which can otherwise be difficult to resolve.
The final analysis step in DNA sequencing, whether done using chain-termination (Proc. Natl. Acad. Sci. U.S.A.; 1977; 74(12): 5463-7 “Sanger sequencing”) or chain-cleavage (Methods-Enzymol.; 1980; 65(1): 499-560) methods, involves the use of a denaturing polyacrylamide electrophoresis gel to separate DNA molecules by size.
Electrophoretic separation based solely on size requires complete elimination of secondary structure from the DNA. This is typically accomplished by using high concentrations of urea in the polyacrylamide matrix and running the gels at elevated temperatures. For most DNAs, this is sufficient to give a regularly spaced pattern of “bands” in a sequencing experiment. Exceptions can occur if the sequence has a dyad symmetry that can form a stable “stem-loop” structure (Proc. Natl. Acad. Sci. U.S.A.; 1987; 84(14): 4767-71). When these sequences are analyzed by gel electrophoresis, some bases may not be completely resolved. The bands representing different bases run at nearly the same position on the gel, “compressed” tightly together, with bands appearing in two or three lanes at the same position. The bases just above the compressed ones are spaced farther apart than normal as well.
Compression artifacts are caused whenever stable secondary structures exist in the DNA under the conditions prevailing in the gel matrix during electrophoresis. The folded structure apparently runs faster through the gel matrix than an equivalent unfolded DNA, catching up with the smaller DNAs in the sequence. Typical gel conditions (7M urea and 50° C.) denature most secondary structures, but not all structures, particularly those with 3 or more G-C base pairs in succession.
Some gel compression artifacts can be eliminated by using an analog of dGTP during synthesis. The use of nucleotide analogs dITP (J. Mol. Biol. 1983; 166: 1-19), 7-deaza-dGTP (Nucleic Acids Res. 1986; 14(3): 1319-24) and 4-aminomethyl dCTP (Nucleic-Acids Res. 1993; 21(11): 2709-14) for dideoxy sequencing have been described. When dITP or 7-deaza-dGTP replace dGTP in the sequencing experiment, the dG nucleotides in the product DNA are replaced by dI or 7-deaza-dG. These form weaker base-pairs with dC, which are more readily denatured for gel electrophoresis. A comparison of the three sequences in FIG. 5 of Proc. Natl. Acad. Sci. U.S.A. 1987; 84(14): 4767-71 shows that dI fully disrupts secondary structure while 7-deaza-dG only partially resolves this relatively strong compression sequence.
None of these substitute nucleotides is adequate for all sequencing situations. For example: 4-aminomethyl dCTP fails to work well with Sequenase DNA polymerase (Nucleic Acids Res. 1993, 21(11): 2709-14); it is difficult to use dITP for sequencing when using thermostable polymerases such as Taq DNA polymerase (Proc. Natl. Acad. Sci. U.S.A. 1988; 85(24): 9436-40); and 7-deaza-dGTP does not resolve all compression sequences (Proc. Natl. Acad. Sci. U.S.A. 1987; 84(14): 4767-71).